Light scattering (LS) is a non-invasive technique for characterizing macromolecules and a wide range of particles in solution. The two types of light scattering detection frequently used for the characterization of macromolecules are static light scattering (SLS) and dynamic light scattering (DLS).
Static light scattering includes a variety of embodiments such as single angle light scattering (SALS), dual angle light scattering (DALS), low angle light scattering (LALS), and multi-angle light scattering (MALS). SLS experiments generally involve the measurement of the absolute intensity of the light scattered from a sample in solution that is illuminated by a fine beam of light. This measurement is often used, for appropriate classes of particles/molecules, to determine the size and structure of the sample molecules or particles, and, when combined with knowledge of the sample concentration, the determination of weight average molar mass. In addition, nonlinearity of the intensity of scattered light as a function of sample concentration may be used to measure interparticle interactions and associations.
Dynamic light scattering is also known as quasi-elastic light scattering (QELS) and photon correlation spectroscopy (PCS). In a DLS experiment, time-dependent fluctuations in the scattered light signal are measured using a fast photodetector. DLS measurements determine the diffusion coefficient of the molecules or particles, which can in turn be used to calculate their hydrodynamic radius.
Extensive literature has been published describing methods for making both static and dynamic light scattering measurements in flowing and batch (non-flowing) systems. See, for example, P. J. Wyatt, “Light scattering and the absolute characterization of macromolecules,” Analytica chimica Acta, 272, 1-40, (1993). Historically light scattering measurements in batch mode were performed on sample solutions contained in scintillation vials. However, these measurements generally required at least 4 mL of the sample. The flow mode alternative might require a lower sample volume within the flow cell itself, however, a significant amount of sample is still required to deliver and fill the measurement volume of the cell. More recently, cuvettes have been introduced with significantly smaller sample volumes. For example, the MicroCUVETTE manufactured by Wyatt Technology Corporation (Santa Barbara, Calif.) is capable of batch LS measurements with sample volumes as small as 12 μL. A common source of small cuvettes are the so-called microtiter plates containing an array of wells, each of which may serve as a cuvette.
As sample volumes decrease, the likelihood of solvent evaporation also becomes a concern and, as light scattering measurements can be very sensitive to the concentration of the solute with the sample, preventing evaporation can become a critical component of accurate LS measurements. It is an objective of the present invention to minimize sample evaporation for batch light scattering measurements. Evaporation can alter the sample state, skew results through altered background intensity, or prevent light scattering measurements entirely. Partial evaporation of the solvent from a cuvette produces an effective increase of the dissolved solute concentration which additionally may have deleterious effects on the sample itself. Evaporation can also impact the meniscus curvature as well as meniscus height in the cuvette or plate well. More substantial evaporation of the sample solvent may prevent any type of LS measurement, a problem particularly prevalent in very small volume cuvettes where even a small amount of evaporation results in a large change in the height of the fluid level. This can cause the meniscus to be intersected by the incident light beam, overwhelming the detector or detectors and distorting significantly any measurements. Further, temperature dependent measurements during which the cuvette environment is cycled over a broad range of ambient temperatures may promote evaporation thus skewing the results and giving inaccurate data regarding temperature dependent reactions, such as molecular interaction experiments.
A frequently used method to overcome the evaporation problem is to cap or place a film over the mouth of the cuvette itself or to place a layer of oil overlaying the sample contained in each well in a microtiter plate. However, for light scattering measurements, both of these commonly used evaporation mitigation techniques can be problematic. With respect to the cap, the sample solvent may still evaporate and condense on the inner surface of the film or cover as the unused volume of the cuvette itself will have a lower vapor pressure than the sample itself, thus evaporation cannot be mitigated by sealing the cuvette alone. While the use of an oil overlay eliminates the issue of condensation, the potentially negative interactions of oil and sample molecules are well known, as documented in the 2004 article by L. S. Jones et al, “Silicone oil induced aggregation of proteins,” published in the Journal of Pharmaceutical Sciences, volume 94, pages 918-927. Such unintended interactions may result in an inaccurate representation of the true sample characteristics. In addition, oil overlays can be difficult to apply, and difficult to remove if the cuvette or well plate is to be reused. It is another objective of the present invention to obviate the need to use an oil overlay to control evaporation in a sample cuvette.
Traditionally, LS measurement cuvettes have been constructed of expensive materials such as quartz and other highly polished glass materials. These cuvettes are often prohibitively expensive to be disposed of after a single use, and therefore must be carefully cleaned between uses to prevent contamination by a prior sample or cleaning agent with the new sample to be analyzed. Further, cuvettes formed from glass substances are rarely capable of being designed in complex forms which would otherwise be useful in providing important benefits to light scattering measurements. It is an objective of the present invention to provide a cuvette compatible with light scattering measurements that is inexpensive enough that it may be discarded after a single use. It is a further objective of this invention to provide a cuvette capable of improved simplicity of use and utility heretofore unavailable in cuvettes made of glass and similar materials.
The limitations disclosed above can seriously inhibit the accurate collection of light scattering data from samples contained within small cuvettes as well as complicating the measurement process by requiring laborious and often inadequate cleaning of the sample containing chamber within the cuvette. A primary objective of the present invention is to provide a means by which all of these limitations for making light scattering measurements in a cuvette may be mitigated, allowing for an increased quality of optical measurements of liquid samples contained therein, while simultaneously facilitating the measurements themselves.